Sample clustering

PCA

##                PC1       PC2        group  condition replicate     name
## WT_4     -32.90151 -5.235742    Control:1    Control         1     WT_4
## WT_5     -28.67289 -8.954453    Control:2    Control         2     WT_5
## WT_6     -41.17100 10.740516    Control:3    Control         3     WT_6
## S926glc4  39.03169 -1.852210 Mutant_LOF:1 Mutant_LOF         1 S926glc4
## S926glc5  24.52983  8.343747 Mutant_LOF:2 Mutant_LOF         2 S926glc5
## S926glc6  39.18388 -3.041858 Mutant_LOF:3 Mutant_LOF         3 S926glc6

Number of genes detected

rRNA percentage

Proportions of gene biotypes

Remove rRNA from the analysis

dds = dds[!(row.names(counts(dds)) %in% ensembl.genes$gene_id[ensembl.genes$gene_biotype %in% c("rRNA", "snoRNA", "snRNA")]),]
dds = dds[rowSums(counts(dds)) > 0,]

Check rRNA removed

Check chrM intact

## 
## chr2L chr2R chr3L chr3R  chr4  chrX  chrY  chrM 
##  2902  3172  2956  3594    91  2326    42    38

Clustering after rRNA removal

PCA after rRNA removal

##                PC1       PC2        group  condition replicate     name
## WT_4     -32.87163 -5.240295    Control:1    Control         1     WT_4
## WT_5     -28.64340 -8.957790    Control:2    Control         2     WT_5
## WT_6     -41.13673 10.755695    Control:3    Control         3     WT_6
## S926glc4  38.99282 -1.833142 Mutant_LOF:1 Mutant_LOF         1 S926glc4
## S926glc5  24.51421  8.298705 Mutant_LOF:2 Mutant_LOF         2 S926glc5
## S926glc6  39.14474 -3.023172 Mutant_LOF:3 Mutant_LOF         3 S926glc6

Size Factors

##      WT_4      WT_5      WT_6  S926glc4  S926glc5  S926glc6 
## 1.0398668 1.0648837 1.1695855 0.8627809 0.9688625 0.9497324

MA Plots

## [1] 1
## [1] 2

## [1] 3

## [1] 2
## [1] 3

## [1] 1
## [1] 2

## [1] 3

## [1] 2
## [1] 3

Normalised counts (boxplot)

seqnames start end width strand gene_id gene_biotype entrezgene_id external_gene_name
FBgn0000559 chr2L 21677913 21682164 4252
FBgn0000559 protein_coding 35422 eEF2
FBgn0013674 chrM 1474 3009 1536
FBgn0013674 protein_coding 192469 mt:CoI
FBgn0013676 chrM 4736 5524 789
FBgn0013676 protein_coding 192471 mt:CoIII
FBgn0013678 chrM 10499 11635 1137
FBgn0013678 protein_coding 192472 mt:Cyt-b
FBgn0284245 chr2R 11891135 11895762 4628
FBgn0284245 protein_coding 36271 eEF1alpha1

These all seem to be protein-coding genes, so we will not remove them.

Normalised counts (Transcripts per million (TPM))

seqnames start end width strand gene_id gene_biotype entrezgene_id external_gene_name
FBgn0066084 chr2R 24903416 24903935 520
FBgn0066084 protein_coding 251466 RpL41

These all seem to be protein-coding genes, so we will not remove them.

Session Info

## R version 4.0.5 (2021-03-31)
## Platform: x86_64-apple-darwin17.0 (64-bit)
## Running under: macOS Big Sur 10.16
## 
## Matrix products: default
## BLAS:   /Library/Frameworks/R.framework/Versions/4.0/Resources/lib/libRblas.dylib
## LAPACK: /Library/Frameworks/R.framework/Versions/4.0/Resources/lib/libRlapack.dylib
## 
## locale:
## [1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8
## 
## attached base packages:
## [1] parallel  stats4    stats     graphics  grDevices utils     datasets 
## [8] methods   base     
## 
## other attached packages:
##  [1] kableExtra_1.3.4            Biostrings_2.58.0          
##  [3] XVector_0.30.0              scales_1.1.1               
##  [5] reshape2_1.4.4              knitr_1.31                 
##  [7] biomaRt_2.46.3              GenomicFeatures_1.42.3     
##  [9] AnnotationDbi_1.52.0        genefilter_1.72.1          
## [11] ggplot2_3.3.3               DESeq2_1.30.1              
## [13] SummarizedExperiment_1.20.0 Biobase_2.50.0             
## [15] MatrixGenerics_1.2.1        matrixStats_0.58.0         
## [17] GenomicRanges_1.42.0        GenomeInfoDb_1.26.7        
## [19] IRanges_2.24.1              S4Vectors_0.28.1           
## [21] BiocGenerics_0.36.0        
## 
## loaded via a namespace (and not attached):
##  [1] nlme_3.1-152             bitops_1.0-6             bit64_4.0.5             
##  [4] webshot_0.5.2            RColorBrewer_1.1-2       progress_1.2.2          
##  [7] httr_1.4.2               rprojroot_2.0.2          tools_4.0.5             
## [10] bslib_0.2.4              utf8_1.2.1               R6_2.5.0                
## [13] mgcv_1.8-34              DBI_1.1.1                colorspace_2.0-0        
## [16] withr_2.4.1              tidyselect_1.1.0         prettyunits_1.1.1       
## [19] bit_4.0.4                curl_4.3                 compiler_4.0.5          
## [22] rvest_1.0.0              xml2_1.3.2               DelayedArray_0.16.3     
## [25] labeling_0.4.2           rtracklayer_1.50.0       sass_0.3.1              
## [28] askpass_1.1              rappdirs_0.3.3           systemfonts_1.0.1       
## [31] stringr_1.4.0            digest_0.6.27            Rsamtools_2.6.0         
## [34] svglite_2.0.0            rmarkdown_2.7            pkgconfig_2.0.3         
## [37] htmltools_0.5.1.1        highr_0.8                dbplyr_2.1.1            
## [40] fastmap_1.1.0            rlang_0.4.10             rstudioapi_0.13         
## [43] RSQLite_2.2.6            farver_2.1.0             jquerylib_0.1.3         
## [46] generics_0.1.0           jsonlite_1.7.2           BiocParallel_1.24.1     
## [49] dplyr_1.0.5              RCurl_1.98-1.3           magrittr_2.0.1          
## [52] GenomeInfoDbData_1.2.4   Matrix_1.3-2             Rcpp_1.0.6              
## [55] munsell_0.5.0            fansi_0.4.2              lifecycle_1.0.0         
## [58] stringi_1.5.3            yaml_2.2.1               zlibbioc_1.36.0         
## [61] plyr_1.8.6               BiocFileCache_1.14.0     grid_4.0.5              
## [64] blob_1.2.1               crayon_1.4.1             lattice_0.20-41         
## [67] splines_4.0.5            annotate_1.68.0          hms_1.0.0               
## [70] locfit_1.5-9.4           pillar_1.6.0             codetools_0.2-18        
## [73] geneplotter_1.68.0       XML_3.99-0.6             glue_1.4.2              
## [76] evaluate_0.14            vctrs_0.3.7              gtable_0.3.0            
## [79] openssl_1.4.3            purrr_0.3.4              assertthat_0.2.1        
## [82] cachem_1.0.4             xfun_0.22                xtable_1.8-4            
## [85] viridisLite_0.4.0        survival_3.2-10          tibble_3.1.0            
## [88] GenomicAlignments_1.26.0 memoise_2.0.0            ellipsis_0.3.1